Novel conjugation reagents

ABSTRACT

The invention provides compound of the general formula: 
     
       
         
         
             
             
         
       
     
     in which each X independently represents a polymer chain; n represents an integer greater than 1; Q represents a linker; Y represents an amide group; and Z represents either —CH.(CH 2 L) 2  or —C(CH 2 L)(═CH 2 ), in which each L independently represents a leaving group. The compounds are useful reagents for the conjugation of polymers to proteins, the resulting conjugates being novel and also forming part of the invention.

This application is a continuation of application Ser. No. 13/919,217,filed Jun. 17, 2013, now allowed; which claims priority benefit ofprovisional Application No. 61/661,300, filed Jun. 18, 2012, andprovisional Application No. 61/720,811, filed Oct. 31, 2012; the entirecontents of which are hereby incorporated by reference in theirentirety.

This invention relates to novel conjugation reagents for conjugatingpolymers to proteins and peptides, and to a novel process for producingnovel conjugates.

Many therapeutically active molecules, for example proteins, do notpossess the properties required to achieve efficacy in clinical medicaluse. For example, many native proteins do not make good medicinesbecause upon administration to patients there are several inherentdrawbacks that include: (1) proteins are digested by many endo- andexo-peptidases present in blood or tissue; (2) almost all proteins areimmunogenic to some extent; and (3) proteins can be rapidly excreted bykidney ultrafiltration and by endocytosis. Some molecules which mightfind utility as active therapeutic agents in medicines are systemicallytoxic or lack optimal bioavailability and pharmacokinetics. Whenproteins clear from the blood circulation quickly they typically have tobe administered to the patient frequently. Frequent administrationfurther increases the risk of toxicity, especially immunologicallyderived toxicities. Often it is difficult to achieve a therapeuticallyeffective dose, so efficacy is compromised. Rapid clearance is thereforeboth an efficacy issue and a safety issue.

Water soluble, synthetic polymers, particularly polyalkylene glycols,are widely used to conjugate therapeutically active molecules such asproteins. These therapeutic conjugates have been shown to alterpharmacokinetics favourably by prolonging circulation time anddecreasing clearance rates, decreasing systemic toxicity, and in severalcases, displaying increased clinical efficacy. The process of covalentlyconjugating polyethylene glycol, PEG, to proteins is commonly known as“PEGylation”.

It is important for optimised efficacy and to ensure dose to doseconsistency that the number of conjugated polymer molecules per proteinis the same for each molecule, and that each polymer molecule isspecifically covalently conjugated to the same amino acid residue ineach protein molecule. Non-specific conjugation at sites along a proteinmolecule results in a distribution of conjugation products and,frequently, unconjugated protein, to give a complex mixture that isdifficult and expensive to purify.

WO 2005/007197 describes a series of novel conjugation reagents whichcan be used to react with nucleophilic groups in a protein to produce aprotein-polymer conjugate. These reagents find particular utility fortheir ability to conjugate with both sulfur atoms derived from adisulfide bond in a protein to give thioether conjugates, and can alsobe used to conjugate with other nucleophiles, for example with twohistidine residues, for example two histidine residues present in apolyhistidine tag attached to a protein, as described in WO 2009/047500.

For some uses, it is desirable to conjugate two polymer chains to aprotein, because the steric properties of a conjugate containing asingle chain of a given molecular weight can be significantly differentfrom the properties of a conjugate containing, for example, two chainseach having half that molecular weight. Reagents capable of suchconjugation are known. Thus for example U.S. Pat. No. 5,932,462 (Harris)discloses reagents capable of conjugating two PEG chains to proteins.Cong et al., Bioconjugate Chemistry 23 (2012) 248-263, also discloses areagent capable of conjugating two PEG chains to proteins, specifically,the PEG-bis-sulfone reagent shown as reagent 3 of FIG. 1, p. 249. InCong's reagent, two PEG chains are attached to different positions on aphenyl group acting as a linker to the functional protein reacting groupof the reagent. Cong's reagent is capable of conjugation of two PEGchains to, for example, two sulfur atoms derived from a disulfide bondin a protein, or two histidine residues present in a polyhistidine tagattached to a protein, which provides improved conjugation compared withthe reagents of Harris.

We have now found a novel reagent capable of conjugating two polymerchains to a protein which shows improved properties over the knownreagent of Cong.

Accordingly, the present invention provides a compound of the generalformula:

in which each X independently represents a polymer chain; n representsan integer greater than 1; Q represents a linker; Y represents an amidegroup; and Z represents either —CH.(CH₂L)₂ or —C(CH₂L)(═CH₂), in whicheach L independently represents a leaving group.

The reagents of the formula I contain at least two polymer chains X,each linked to linker Q. Each polymer X may for example be apoly(alkylene glycol), a polyvinylpyrrolidone, a polyacrylate, forexample poly(acryloyl morpholine), a polymethacrylate, a polyoxazoline,a polyvinylalcohol, a polyacrylamide or polymethacrylamide, for examplepolycarboxymethacrylamide, or a HPMA copolymer. Additionally the polymermay be one that is susceptible to enzymatic or hydrolytic degradation.Such polymers, for example, include polyesters, polyacetals, poly(orthoesters), polycarbonates, poly(imino carbonates), and polyamides, such aspoly(amino acids). A polymer may be a homopolymer, random copolymer or astructurally defined copolymer such as a block copolymer. For example itmay be a copolymer, e.g., a block copolymer, derived from two or morealkylene oxides, or from poly(alkylene oxide) and either a polyester,polyacetal, poly(ortho ester), or a poly(amino acid). The so-calledPluronics are an important class of PEG block copolymers. These arederived from ethylene oxide and propylene oxide blocks. Polyfunctionalpolymers that may be used include copolymers of divinylether-maleicanhydride and styrene-maleic anhydride.

Naturally occurring polymers may also be used, for examplepolysaccharides such as chitin, dextran, dextrin, chitosan, starch,cellulose, glycogen, poly(sialylic acid) and derivatives thereof. Aprotein may be used as the polymer. This allows conjugation of oneprotein, for example an antibody or antibody fragment, to a secondprotein, for example an enzyme or other active protein. Also, if apeptide containing a catalytic sequence is used, for example an O-glycanacceptor site for glycosyltransferase, it allows the incorporation of asubstrate or a target for subsequent enzymatic reaction. Poly(aminoacid)s such as polyglutamic acid or polyglycine may also be used, as mayhybrid polymers derived from natural monomers such as saccharides oramino acids and synthetic monomers such as ethylene oxide or methacrylicacid.

Preferably each polymer used in the present invention is a hydrophilicor water-soluble, synthetic polymer. If a polymer is a poly(alkyleneglycol), this is preferably one containing C₂ and/or C₃ units, and isespecially a poly(ethylene glycol) (PEG). Except where the contextrequires otherwise, any reference to a polymer in this specificationshould be understood to include a specific reference to PEG.

A polymer may optionally be derivatised or functionalised in any desiredway. Reactive groups may be linked at the polymer terminus or end group,or along the polymer chain through pendent linkers; in such cases, thepolymer is for example a polyacrylamide, polymethacrylamide,polyacrylate, polymethacrylate, or a maleic anhydride copolymer. Suchfunctionalised polymers provide a further opportunity for preparingmultimeric conjugates (i.e., conjugates in which the polymer isconjugated to more than one molecule). For example, a polymer may carryone or more drug molecules at any point along its length, for example atits terminus. If desired, the polymer may be coupled to a solid supportusing conventional methods.

The two or more polymer chains X may be the same or different.Specifically, each X may represent the same chemical polymer, or adifferent chemical polymer. For example, each X may represent a PEGchain, or one X may represent a PEG chain and another X may represent adifferent polymer, for example a PVP or a protein chain.

Each polymer X may contain a single linear chain, or it may havebranched morphology composed of many chains either small or large.Generally, a polymer chain is initiated or terminated by a suitable endgroup, and is connected at the other end of the chain to the linkergroup Q: for example, a PEG chain may have an end group selected fromalkoxy, e.g., methoxy, aryloxy, carboxy or hydroxyl. Where the chain isbranched, each free branch terminus will carry the end group. Whenpreparing the reagents of the invention, the other end of the polymer Xwill be reacted with a compound containing the linker Q, and the natureof this linkage depends upon chemical convenience. For example, when thepolymer is PEG, the terminal —OH group may be reacted with a suitablecomplementary group on Q. Alternatively, as is well known in the art,the PEG can be converted to the PEG amine, PEG-NH₂, which tends to bemore reactive than PEG alcohol, before reaction with a suitablecomplementary group on Q. Thus, some PEG-containing reagents accordingto the invention will include an oxygen atom linking PEG to Q, whileothers will include an NH group linking PEG to Q, for exampleCH₃O—(CH₂CH₂O)_(m)— or CH₃O—(CH₂CH₂O)_(m)—(CH₂CH₂NH)— in which m is thenumber of ethylene oxide units in the PEG, and in which the end group isshown as methoxy for convenience.

Each polymer chain X may have any suitable molecular weight, and eachpolymer chain X may have the same or different molecular weight as anyother. For example each chain may have a molecular weight of at least 5,10, 15, 20, 30, or 40 kDa. Generally, the preferred maximum molecularweight of each chain is 60 kDa. When a conjugate is intended to leavethe circulation and penetrate tissue, for example for use in thetreatment of inflammation caused by malignancy, infection or autoimmunedisease, or by trauma, it may be advantageous to use a conjugate inwhich the total molecular weight of the polymers (X)_(n) is in the range2000-30,000 g/mol. For applications where the conjugate is intended toremain in circulation it may be advantageous to use a higher totalmolecular weight of polymer, for example in the range of 20,000-75,000g/mol.

n must be greater than 1, for example up to 4, for example 2 or 3.Reagents and conjugates in which n is 2 are particularly preferred.

The reagents of the present invention contain an amide group, Y, whichas drawn in the formula I may be —CO—NR′— or, preferably, —NR′—CO—, inwhich R′ represents a C₁₋₄ alkyl group, for example a methyl group, or,especially, a hydrogen atom. This group may be linked to the phenylgroup shown in formula I at any position, but is preferably in the paraposition relative to the —CO.Z group. The phenyl group of the formula Imay carry additional substituents if desired, but is preferablyunsubstituted.

In order to carry more than one polymer chain, the linker must containat least one branching atom, generally a carbon or a nitrogen atom.Where the branching atom is a carbon atom, a linking group Q may forexample be an alkylene group (preferably a C₁₋₁₀ alkylene group) or anoptionally-substituted aryl (for example phenyl) or heteroaryl group,any of which may be terminated or interrupted and/or terminated by oneor more oxygen atoms, sulfur atoms, —NR groups (in which R represents ahydrogen atom or an alkyl (preferably C₁₋₆alkyl), aryl (preferablyphenyl), or alkyl-aryl (preferably C₁₋₆alkyl-phenyl) group), ketogroups, —O—CO— groups, —CO—O— groups, —O—CO—O, —O—CO—NR—, —NR—CO—O—,—CO—NR— and/or —NR.CO— groups. Preferably the linker is a C₁₋₁₀ alkylenegroup, especially a C₁₋₆ alkylene group, interrupted and/or terminatedby one or more oxygen atoms and/or NH groups and/or keto groups,especially a C₁₋₆alkylene group interrupted by an oxygen atom. Anespecially preferred reagent of the formula I contains a linker group Qof formula:

in which p is 1 to 6, for example 1, 2 or, especially, 3.

Another specific group of reagents of the formula I contains a linkergroup Q of formula:

in which p has the meaning given above. When p is 1, this linker isderived from aspartic acid, and when p is 2, this linker is derived fromglutamic acid.

In another embodiment of the invention, the branching atom may benitrogen, and the linker may be one of those mentioned above in whichthe branching carbon atom is replaced by a branching nitrogen atom. Forexample, in one specific embodiment of the invention, the linker may beof the formula:

where a linker group Q is terminated adjacent the amide group Y by anoxygen atom or an NH group, then the combination of this terminatinggroup and Y forms a urethane or urea group —O—CO—NR′— or —NH—CO—NR′—.

The linkage via the linker Q to the polymer may be by way of ahydrolytically labile bond, or by a non-labile bond.

A leaving group L may for example represent —SR, —SO₂R, —OSO₂R, —N⁺R₃,—N⁺HR₂, —N⁺H₂R, halogen, or —OØ, in which R has the meaning given above,and Ø represents a substituted aryl, especially phenyl, group,containing at least one electron withdrawing substituent, for example—CN, —NO₂, —CO₂R, —COH, —CH₂OH, —COR, —OR, —OCOR, —OCO₂R, —SR, —SOR,—SO₂R, —NHCOR, —NRCOR, —NHCO₂R, —NRCO₂R, —NO, —NHOH, —NROH, —C═N—NHCOR,—C═N—NRCOR, —N⁺R₃, —N⁺HR₂, —N⁺H₂R, halogen, for example chlorine or,especially, bromine or iodine, —C≡CR, —C═CR₂ and —C═CHR, in which each Rindependently has one of the meanings given above. Alkyl or arylsulfonyl groups are particularly preferred leaving groups, withphenylsulfonyl or, especially, tosyl, being especially preferred. Wheretwo Ls are present, these may be different groups, but preferably theyare the same group.

Except where otherwise stated, substituents which may be present on anyoptionally substituted aryl, for example phenyl, or heteroaryl grouppresent in a compound of formula I include for example one or more ofthe same or different substituents selected from alkyl (preferably C₁₋₄alkyl, especially methyl, optionally substituted by OH or CO₂H), —CN,—NO₂, —CO₂R, —COH, —CH₂OH, —COR, —OR, —OCOR, —OCO₂R, —SR, —SOR, —SO₂R,—NHCOR, —NRCOR, NHCO₂R, —NR.CO₂R, —NO, —NHOH, —NR.OH, —C═N—NHCOR,—C═N—NR.COR, —N⁺R₃, —N⁺H₃, —N⁺HR₂, —N⁺H₂R, halogen, for example fluorineor chlorine, —C≡CR, —C═CR₂ and —C═CHR, in which each R independently hasone of the meanings given above. Preferred substituents, if present,include for example CN, NO₂, —OR, —OCOR, —SR, —NHCOR, —NR.COR, —NHOH and—NR.COR.

Especially preferred reagents according to the invention have theformulae:

In these reagents, preferably X is polyethylene glycol, especiallymethoxy-terminated polyethylene glycol, i.e., CH₃O—(CH₂CH₂O)_(m)— inwhich m is the number of ethylene oxide units in the PEG. In addition,in these reagents, preferably each L is a tosyl group, thus:

The compounds of formula I may be used for conjugation to a protein orpeptide. For convenience, the term “protein” will be used throughoutthis Specification, and except where the context requires otherwise, theuse of the term “protein” should be understood to include a reference topeptide.

Accordingly, the invention further provides a process for thepreparation of a polymer conjugate, which comprises reacting a compoundof the general formula I with a protein or a peptide. The resultingconjugates have the general formula:

in which X, n, Q and Y have the meanings given above, and either each ofPr¹ and Pr² represents a separate protein or peptide molecule, or Pr¹and Pr² together represent a single protein or peptide Pr bonded at twoseparate points, thus:

Preferably Pr¹ and Pr² together represent a single protein bonded to twosulfur atoms derived from a disulfide bond in a protein, or to twohistidine residues present in a polyhistidine tag attached to a protein(i.e., the resulting conjugates have the general formula IV(a)).

In the reagent of formula I, Z represents either —CH.(CH₂L)₂ or—C(CH₂L)(═CH₂). These two groups are chemically equivalent to eachother. If a reagent of formula I in which Z represents —CH.(CH₂L)₂,i.e., a reagent of formula Ia:

is used to react with a protein in a process according to the invention,the reaction proceeds by the loss of one leaving group L, and resultantformation of a reagent of formula I in which Z represents—C(CH₂L)(═CH₂), i.e., a reagent of formula 1b:

This reagent reacts with one nucleophile, for example a cysteine,histidine or lysine residue, in the protein. Subsequently, the remainingleaving group L is lost, and reaction with a second nucleophile (eitherin a second molecule of protein or in the same protein molecule as thefirst nucleophile) occurs to form the desired conjugate. Therefore, theprocess of the invention can be carried out using a compound of formula1a as a starting material, in which case a compound of formula Ib isformed in situ, or a pre-formed compound of formula Ib may be used asstarting material.

The conjugation reaction according to the invention may be carried outunder the reaction conditions described in WO 2005/007197 and WO2009/047500. The process may for example be carried out in a solvent orsolvent mixture in which all reactants are soluble. For example, theprotein may be allowed to react directly with the polymer conjugationreagent in an aqueous reaction medium. This reaction medium may also bebuffered, depending on the pH requirements of the nucleophile. Theoptimum pH for the reaction will generally be at least 4.5, typicallybetween about 5.0 and about 8.5, preferably about 6.0 to 7.5. Theoptimal reaction conditions will of course depend upon the specificreactants employed.

Reaction temperatures between 3-37° C. are generally suitable when usingan aqueous reaction medium. Reactions conducted in organic media (forexample THF, ethyl acetate, acetone) are typically conducted attemperatures up to ambient.

Where bonding to the protein is via two sulfur atoms derived from adisulfide bond in the protein, the process may be carried out byreducing the disulfide bond in situ following which the reduced productreacts with the reagent of the formula I. Preferably the disulfide bondis reduced and any excess reducing agent is removed, for example bybuffer exchange, before the conjugation reagent is introduced. Thedisulfide can be reduced, for example, with dithiothreitol,mercaptoethanol, or tris-carboxyethylphosphine using conventionalmethods.

The protein can be effectively conjugated using a stoichiometricequivalent or a slight excess of conjugation reagent I. However, it isalso possible to conduct the conjugation reaction with an excessstoichiometry of conjugation reagent, and this may be desirable for someproteins. The excess reagent can easily be removed, for example by ionexchange chromatography, during subsequent purification of theconjugate.

Compounds of the general formula I in which Z represents —CH.(CH₂L)₂ maybe prepared by either reacting a compound of the general formula

(X)_(n)-Q-NH₂  (III)

with a compound of the general formula

or by reacting a compound of the general formula

(X)_(n)-Q-CO₂H  (V)

with a compound of the general formula

In both cases, an amide group is formed. As is well known in the art,the CO₂H group which is reacted to form the amide group, is suitablyactivated to facilitate the reaction, for example by formation of anactive ester, an acyl chloride, or an anhydride, or directly with amineby the use of an activation agent such as a carbodiimide.

As explained above, compounds of the general formula I in which Zrepresents —C(CH₂L)(═CH₂) may be prepared by removing a leaving group Lfrom the corresponding compound of the general formula I in which Zrepresents a —CH.(CH₂L)₂ group.

The immediate product of the process according to the invention is aconjugate which still contains the keto group CO attached to the phenylring in formula I, i.e., a conjugate of formula II, especially IIa,above. However, the process of the invention is reversible undersuitable conditions. This may be desirable for some applications, forexample where rapid release of the protein is required, but for otherapplications, rapid release of the protein may be undesirable. It maytherefore be desirable to stabilise the conjugates by reduction of theketo group to give a moiety which prevents release of the protein,typically a hydroxyl group OH, although reductive amination is also apossibility, giving an amine group CH.NH₂, CH.NHR or CH.NR₂ in whicheach R independently has the meaning given above. These groups may befurther reacted if desired, for example a hydroxy group may be convertedinto an ether group CH.OR by reaction with an etherifying agent; anester group CH.O.C(O)R may be obtained by the reaction of a hydroxygroup with an acylating agent; or an amide CH.NHC(O)R or CH.N(C(O)R)₂may be formed by acylation of an amine. Accordingly, the processaccording to the invention may comprise the additional step of reducingthe keto group in the conjugate. The use of a borohydride, for examplesodium borohydride, sodium cyanoborohydride, potassium borohydride orsodium triacetoxyborohydride, as reducing agent is particularlypreferred. Other reducing agents which may be used include for exampletin(II) chloride, alkoxides such as aluminium alkoxide, and lithiumaluminium hydride.

Conjugates preparable by the process of the present invention are novel,and therefore form part of the present invention per se. Novelconjugates according to the present invention have the general formula:

in which X, n, Q and Y have the meanings given above, A represent agroup CO, CHOH, CH.NH₂, CH.NHR, CH.NR₂, CH.OR CH.O.C(O)R, CH.NHC(O)R, orCH.N(C(O)R)₂, in which each R has the meaning given above, and eithereach of Pr¹ and Pr² represents a separate protein or peptide molecule orboth of Pr¹ and Pr² together represent a single protein or peptidebonded at two separate points. Preferred conjugates have the generalformula:

in which X, n, Q, Y and A have the meanings given above, and Prrepresents a single protein or peptide bonded at two separate points.

Especially preferred conjugates are of the formulae:

Of course, it is possible for more than one conjugating reagent of theformula I to be conjugated to a protein, where the protein containssufficient suitable attachment points. For example, in a protein whichcontains two different disulfide bonds, or in a protein which containsone disulfide bond and also carries a polyhistidine tag, it is possibleto conjugate two molecules of the reagent of formula I per molecule ofprotein.

Suitable proteins which may be conjugated using the process of theinvention include for example peptides, polypeptides, antibodies,antibody fragments, enzymes, cytokines, chemokines, receptors, bloodfactors, peptide hormones, toxin, transcription proteins, or multimericproteins.

The following gives some specific proteins which may be conjugated usingthe present invention. Enzymes include carbohydrate-specific enzymes,proteolytic enzymes and the like, for example the oxidoreductases,transferases, hydrolases, lyases, isomerases and ligases disclosed byU.S. Pat. No. 4,179,337. Specific enzymes of interest includeasparaginase, arginase, adenosine deaminase, superoxide dismutase,catalase, chymotrypsin, lipase, uricase, bilirubin oxidase, glucoseoxidase, glucuronidase, galactosidase, glucocerbrosidase, glucuronidase,and glutaminase.

Blood proteins include albumin, transferrin, Factor VII, Factor VIII orFactor IX, von Willebrand factor, insulin, ACTH, glucagen, somatostatin,somatotropins, thymosin, parathyroid hormone, pigmentary hormones,somatomedins, erythropoietin, luteinizing hormone, hypothalamicreleasing factors, antidiuretic hormones, prolactin, interleukins,interferons, for example IFN-α or IFN-β, colony stimulating factors,hemoglobin, cytokines, antibodies, antibody fragments,chorionicgonadotropin, follicle-stimulating hormone, thyroid stimulatinghormone and tissue plasminogen activator.

Other proteins of interest are allergen proteins disclosed by Dreborg etal., Crit. Rev. Therap. Drug Carrier Syst. 6 (1990) 315-365 as havingreduced allergenicity when conjugated with a polymer such aspoly(alkylene oxide) and consequently are suitable for use as toleranceinducers. Among the allergens disclosed are Ragweed antigen E, honeybeevenom, mite allergen and the like.

Glycopolypeptides such as immunoglobulins, ovalbumin, lipase,glucocerebrosidase, lectins, tissue plasminogen activator andglycosilated interleukins, interferons and colony stimulating factorsare of interest, as are immunoglobulins such as IgG, IgE, IgM, IgA, IgDand fragments thereof.

Of particular interest are receptor and ligand binding proteins andantibodies and antibody fragments which are used in clinical medicinefor diagnostic and therapeutic purposes. The antibody may be used aloneor may be covalently conjugated (“loaded”) with another atom or moleculesuch as a radioisotope or a cytotoxic/antiinfective drug. Epitopes maybe used for vaccination to produce an immunogenic polymer-proteinconjugate.

Particularly preferred proteins include antibody fragments, for exampleIgG Fab fragment, and interferons, such as IFN-α, IFN-β and consensusIFN.

The protein may be derivatised or functionalised if desired. Inparticular, prior to conjugation, the protein, for example a nativeprotein, may have been reacted with various blocking groups to protectsensitive groups thereon; or it may have been previously conjugated withone or more polymers or other molecules, either using the process ofthis invention or using an alternative process. In one preferredembodiment of the invention, it contains a polyhistidine tag, which canbe targeted by the conjugation reagent according to the invention.

The invention further provides a pharmaceutical composition comprising aconjugate according to the invention together with a pharmaceuticallyacceptable carrier, and optionally also containing a further activeingredient in addition to the conjugate according to the invention; aconjugate according to the invention for use in therapy; the use of aconjugate according to the invention in a process for the manufacture ofa medicament; and a method of treating a patient which comprisesadministering a pharmaceutically-effective amount of a conjugate or apharmaceutical composition according to the invention to a patient.

The conjugating reagents of the present invention have been found to beextremely useful, being capable of highly efficient site-specificconjugation to proteins, the resulting novel conjugates demonstrating ahigh level of stability. As illustrated in the examples below,dramatically improved efficiency over the comparable known reagent ofCong et al., Bioconjugate Chemistry 23 (2012) 248-263, is obtained.

The accompanying drawings illustrate results obtained in the followingExamples:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the SDS-PAGE gel obtained in Example 2.

FIGS. 2, 3 and 4 illustrate the SDS-PAGE gels obtained in Example 3.

FIG. 5 illustrates the SDS-PAGE gel obtained in Example 4.

FIG. 6 illustrates the SDS-PAGE gels obtained in Example 7.

The following Examples illustrate the invention.

EXAMPLE 1: PREPARATION OF PEG REAGENT 1

40 (2×20) kDa bifurcated PEG amine 3 (MPEG being CH₃O.CH₂CH₂O)_(m)—) waspurchased from NOF CORPORATION (SUNBRIGHT GL2-400 PA, lot: M7D902).4-[2,2-bis[(p-tolylsulfonyl) methyl] acetyl]benzoic acid-NHS ester, 4was prepared according to Brocchini et al., Nat. Protoc. 1 (2006)2241-2252.

To a single neck round-bottomed flask containing a magnetic stirrer bar,was added bifurcated PEG amine 3 (300 mg) and toluene (8 mL). Theresulting homogeneous solution was evaporated under reduced pressureusing a rotary evaporator for 2 h to leave a solid residue. The residuewas dissolved in dichloromethane (15 mL), the flask was sealed with aseptum and the mixture stirred under argon. To the solution was addedactivated linker 4 (27 mg), the flask was resealed with a septum and thereaction was stirred at rt overnight. The septum was removed and thevolatile portion was removed via evaporation under reduced pressureusing a rotary evaporator. Acetone (20 mL) was added to the residue andthe solid was dissolved with gentle warming (30° C.). The resultingsolution was filtered through non-absorbent cotton wool into a 50 mLFalcon tube. Cooling the solution in a dry-ice bath resulted in a thickprecipitate. Centrifugation (−9° C., 4000 rpm) for 30 min sedimented theprecipitate. The supernatant was decanted and the pellet was againdissolved in acetone (20 mL) at 30° C. Precipitation, sedimentation anddecanting were performed as previously described. A third cycle ofacetone precipitation and sedimentation was performed and afterdecanting the supernatant, the pellet was frozen at −80° C. and thendried to constant mass under high vacuum to give PEG reagent 1 as anoff-white solid (227 mg). ¹H NMR (CDCl₃): δ (ppm) 2.49 (s, 6H), 3.38 (s,6H), 3.45-3.86 (m), 4.33 (m, 1H), 7.36 (AA‘BB’, 4H), 7.64 (AA‘BB’, 2H),7.68 (AA‘BB’, 4H), 7.83 (AA‘BB’, 2H).

EXAMPLE 2: COMPARISON OF THE REACTIVITY OF PEG REAGENTS 1 AND 2 WITH AHUMAN IGG, FAB Fragment

In this example, PEG reagent 1 of Example 1 was compared with thefollowing reagent, PEG reagent 2, in which MPEG isCH₃O.(CH₂CH₂O)_(m-1)—CH₂CH₂—, of Cong et al., supra.:

A human IgG, Fab fragment solution (4.0 mg, 0.909 mL) JacksonImmunoResearch Laboratories Inc. Cat. No. 009-000-007) was diluted to4.95 mL with 50 mM sodium phosphate, pH 7.4 (containing 150 mM NaCl and20 mM EDTA). To the Fab fragment solution, 1.0 M DTT (50 μL) was addedto give a final DTT concentration of 10 mM in order to reduce theinterchain disulfide bond so that PEGylation could occur. The resultingsolution was mixed gently and then stood at 4° C. for 1 h. The solutionof reduced Fab was buffer exchanged into 50 mM sodium phosphate, pH 7.4(containing 150 mM NaCl and 20 mM EDTA) using PD-10 desalting columns.The reduced Fab solution was split equally into two portions (3.5 mL, ˜2mg). Two PEG reagents: PEG reagent 1 and PEG reagent 2, were dissolvedin 50 mM sodium phosphate, pH 7.4 (containing 150 mM NaCl and 20 mMEDTA) at 20 mg/mL. To the first portion of Fab solution, PEG reagent 1(75 μL, 1.5 mg) was added and to the second portion of Fab solution, PEGreagent 2 (75 μL, 1.5 mg) was added. Both reactions were mixed gentlyand then stood at 4° C. for 20 h. After 20 h the crude reaction mixtureswere analysed by SDS-PAGE. The gel was stained with INSTANTBLUE™ andimaged using an IMAGEQUANT™ LAS 4010 instrument. The result is shown inFIG. 1. In FIG. 1, lane M indicates Novex Protein Standards; lane 1indicates human IgG, Fab fragment; lane 2 indicates PEGylated productfrom PEG reagent 2 at 20 h; and lane 3 indicates PEGylated product fromPEG reagent 1 at 20 h. From the SDS-PAGE analysis it can be seen that,while both PEG reagents 1 and 2 were successfully conjugated to the Fabfragment, the efficiency of the conjugation for PEG reagent 1 was 26%,over double of that for PEG reagent 2 (10%).

EXAMPLE 3: STABILITY COMPARISONS OF IFN α-2A CONJUGATES PREPARED WITHPEG REAGENTS 1 and 2

Preparation of Conjugates:

A solution of IFN α-2a (6.5 mg, 0.845 mg/mL) was prepared in 50 mMsodium phosphate buffer, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA).The protein solution was diluted with buffer (313 L) and a 1.0 mM DTTsolution in water (187.5 mL) was then added to give a final DTTconcentration of 25 mM and a reaction volume of 7.5 mL. After gentlemixing, the reaction was stood at room temperature for 30 min. Thereduced protein was buffer exchanged into 50 mM sodium phosphate, pH 7.4(containing 150 mM NaCl and 20 mM EDTA) using PD-10 columns. The elutedprotein solution was centrifuged (3000 g, 4° C., 5 min) and thesupernatant was then quantified by UV absorbance measurements at 280 nm(0.532 mg/mL). The protein solution was diluted to 0.10 mg/mL withbuffer. PEGs 1 and 2 were dissolved at 20 mg/mL in 50 mM sodiumphosphate, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA). Two vialswere each charged with reduced IFN α-2a (2.5 mg, 24.8 mL); to the firstvial PEG reagent 1 (4.9 mg, 0.245 mL) was added and to the second vialPEG reagent 2 (4.9 mg, 0.245 mL) was added. The reactions were mixedgently and then stood at 4° C. for 18 h. Any reduced protein in thefinal reaction mixtures was oxidised by sequentially adding 5 mg/mLcopper sulfate (12.18 μL) and then 50:50 (mM) GSH/GSSG (0.25 mL). Thereoxidation reaction was conducted at 4° C. overnight. The reactionmixtures were diluted ×4 with 100 mM sodium acetate, pH 4 and thenpurified by cation exchange chromatography (MACROCAP™ SP) using a stepgradient elution of 100 mM sodium acetate, pH 4 (1.0 M NaCl) with thedesired conjugates eluting at 0.60-0.65 M NaCl.

Stability Comparison 1. Stress Tests for IFN α-2a Conjugates Preparedwith PEG Reagents 1 and 2:

For each of the IFN α-2a samples PEGylated with 1 and 2 (in filtersterilised PBS), four vials were prepared. Each vial was loaded with 20μL of conjugate at a concentration of 200 μg/mL. Two vials for each ofthe test samples contained 10 mM DTT. One vial with and one vial withoutDTT were heated to 50° C. for 1 h. The remaining vials were heated to90° C. for 10 min. The samples (along with unconjugated protein andunstressed conjugate) were analysed by SDS-PAGE—gels were stained withINSTANTBLUE™ and imaged using an IMAGEQUANT™ LAS 4010 instrument and theresults are shown in FIGS. 2 and 3 for PEG 1-IFN α-2a and PEG 2-IFN α-2arespectively. In FIGS. 2 and 3, lane M indicates Novex ProteinStandards; lane 1 indicates IFN α-2a; lane 2 indicates PEG-IFN α-2a;lane 3 indicates PEG-IFN α-2a −50° C., 1 h; lane 4 indicates PEG-IFNα-2a −50° C., DTT, 1 h; lane 5 indicates PEG-IFN α-2a −90° C., DTT, min;and lane 6 indicates PEG-IFN α-2a −90° C., DTT, 10 min.

FIGS. 2 and 3 show that both of the conjugates tested were stable at 50°C. for 1 h. However, after heating at 50° C. for 1 h in the presence of10 mM DTT, significantly more free protein and aggregation is observedfor the conjugate prepared with PEG reagent 2. Thermal stress at 90° C.for 10 min resulted in release of free protein for the conjugatePEGylated with 2 but not for the conjugate PEGylated with 1.

Stability Comparison 2. 28 Day, 40° C., Accelerated Stability Studiesfor IFN α-2a Conjugates PEGylated with PEG Reagents 1 and 2.

Solutions of the two test samples were made up in filter sterilised PBS(containing 0.01% (w/v) NaN₃) at a protein concentration of 200 μg/mL.For each of PEG 1-IFN α-2a and PEG 2-IFN α-2a four vials were loadedwith 100 μL of test sample. One vial for each sample was immediatelyfrozen at −80° C. (t=0 days). The remaining three were sealed withPARAFILM® and then were stored at 40° C. At 7, 14 and 28 days a samplewas removed from storage and frozen at −80° C. until the completion ofthe study. The test samples were flash thawed in a water baththermostated at 37° C. and analysed by SDS-PAGE (INSTANTBLUE™ stainedand imaged using an IMAGEQUANT™ LAS 4010 instrument) and the result isshown in FIG. 4, in which lane M indicates Novex Protein Standards; lane1 indicates IFN α-2a (1 μg); lane 2 indicates PEG 2-IFN α-2a, Day 0;lane 3 indicates PEG 2-IFN α-2a, Day 7; lane 4 indicates PEG 2-IFN α-2a,Day 14; lane 5 indicates PEG 2-IFN α-2a, day 28; lane 6 indicates PEG1-IFN α-2a, Day 0; lane 7 indicates PEG 1-IFN α-2a, Day 7; lane 8indicates PEG 1-IFN α-2a, Day 14; lane 6 indicates PEG 1-IFN α-2a, Day28. In FIG. 4, it can clearly be seen that the PEG 2-IFN α-2a is lessstable than PEG 1-IFN α-2a with more free protein and less conjugateremaining at each time point.

EXAMPLE 4: CONJUGATION OF IFN-β-1B WITH PEG REAGENT 1

To disulfide reduced IFN-β-1b (9.5 mg, 0.3 mg/mL) at pH 7.3 was added asolution of PEG reagent 1 (1.7 mL, 20 mg/mL) at pH 7.3. The resultingsolution was allowed to incubate at 22° C. for 4 h, whereupon the crudereaction mixture was analysed by SDS-PAGE. The gel was stained withINSTANTBLUE™ and imaged using an IMAGEQUANT™ LAS 4010 instrument. Theresult is shown in FIG. 5. In FIG. 5, in lane M are Novex proteinstandards; lane 1 is the starting IFN-β-1b; lane 2 is the reducedIFN-β-1b and lane 3 is the reaction mixture of PEG reagent 1 withIFN-β-1b. From the SDS-PAGE analysis it can be seen that PEGylation ofIFN-β-1b occurred successfully with a product band visible level withthe 110 kDa protein standard.

EXAMPLE 5: PREPARATION OF BRANCHED PEG REAGENT 11

Step 1: Derivatisation of O-(2-aminoethyl)-O′-methyl (polyethyleneglycol) with Fmoc-L-aspartic acid 4-tert-butyl ester

To a single neck round-bottom flask was added Fmoc-L-aspartic acid4-tert-butyl ester 5 (16.46 mg) dissolved into anhydrous dichloromethane(DCM) (5 mL). Fresh N,N′-diisopropylcarbodiimide (DIPC) (6.3 μL) wasadded and the reaction mixture was allowed to stir, at room temperaturefor 30 min. O-(2-aminoethyl)-O′-methyl (polyethylene glycol) 10 (100 mg)was placed separately in a one neck 50 mL schlenk flask fitted with aseptum and a magnetic stir bar. Azeotropic distillation was carried outusing 5 mL of toluene with the aid of an oil pump fitted with an icetrap to dry the polymer prior to the coupling reaction. Anhydrous DCM (5mL) was added to the flask under argon atmosphere to dissolve the PEGcompletely. 4-Dimethylaminopyridine (DMAP) (1.22 mg) was added to thePEG solution. The activated aspartic derivative was injected into thePEG solution drop-wise. The resulting solution was allowed to stir for48 h at room temperature under argon atmosphere. After this time,volatiles were removed by roto-evaporation and the crude product wasplaced under vacuum for 40 min. The crude was then re-dissolved inacetone and filtered through cotton-wool into a pre-weighed centrifugetube. The sample was placed in dry-ice for precipitation to occur. Theprecipitate was isolated by centrifugation (−9° C., 4000 rpm, 30 min).The solid obtained was dried in a desiccator to afford a white-offproduct (89 mg) ¹H-NMR (CDCl₃, 400 MHz) δ/ppm: 1.45 (s, 9H), 2.6-2.8(dd, 2H), 3.38 (s, 3H), 3.5-3.8 (broad, PEG), 4.22 (t, 1H), 4.41 (d,2H), 4.50 (m, 1H), 5.94 (br, 1H), 6.79 (br, 1H), 7.31 (t, 2H), 7.40 (t,2H), 7.56 (d, 2H), 7.77 (d, 2H)

Step 2: Removal of Fluorenylmethyloxycarbonyl from 6

Aspartic PEG derivative 6 (50 mg) was dissolved in dimethylformamide(DMF) (0.8 mL) in a single neck round-bottom flask (Kocienski, 1994).Under magnetic stirring, a solution of piperidine (0.2 mL) was addeddrop-wise. The reaction mixture was allowed to stir at room temperaturefor 10 min. After this time, volatiles were removed by roto-evaporationand the crude product was dried under vacuum for 1 hour. The crude wasthen purified by the dry-ice precipitation method described in step 1. Awhite product was afforded after drying in a desiccator (44 mg). ¹H-NMR(CDCl₃, 400 MHz) δ/ppm: 1.36 (s, 9H), 2.76-2.49 (dd, 2H), 3.31 (s, 3H),3.5-3.8 (broad, PEG), 4.31 (t, 1H), 6.89 (br, 1H)

Step 3: Preparation of Mono-PEG Aspartic Derivative DB Reagent 8

To a single neck round-bottom flask was added aspartic acid 4-tert-butylester PEG derivative 7 (90 mg) along with 5 mL of toluene, forazeotropic distillation. After complete dryness, anhydrous DCM (5 mL)was added to the flask under argon atmosphere to dissolve 7 completely.4-Dimethylaminopyridine (DMAP) (1.1 mg) was added to the solution. In aseparate flask, the disulfide-bridging linker (15.7 mg) was added anddissolved into anhydrous DCM (5 mL). Fresh N,N′-diisopropylcarbodiimide(DIPC) (4.5 mg, 5.31 μL) was added and the reaction mixture was allowedto stir, at room temperature for 30 min. The activated linker was thenadded to the PEG solution drop-wise. After a total reaction time of 48h, volatiles are removed by roto-evaporation and the crude product wasplaced under vacuum for 40 min. Purification and isolation of productwas achieved by the dry-ice precipitation method described in sectionStep 1. The solid obtained was dried in a desiccator to afford awhite-off product (83.5 mg). ¹H-NMR (CDCl₃, 400 MHz) δ/ppm: 1.43 (s,9H), 2.33 (s, 6H), 2.62-2.91 (dd, 2H), 3.14-3.22 (m, 4H), 3.36 (s, 3H),3.5-3.8 (br, PEG), 4.89 (t, 1H), 6.97 (br, 1H), 7.05 (d, 4H), 7.12 (d,4H), 7.56 (d, 2H), 7.73 (d, 2H)

Step 4: Boc Group Removal from Mono PEG DB Aspartic Derivative 8

Aspartic PEG derivative 8 (50 mg) was dissolved in anhydrous DCM (0.9mL) in a single neck round-bottom flask. Under magnetic stirring, asolution of trifluoroacetic acid (0.1 mL) was added drop-wise(Kocienski, 1994). The reaction mixture was allowed to stir at roomtemperature for 2 h. After this time, volatiles were removed byroto-evaporation and the crude product was dried under vacuum for 1 h.Isolation of product was achieved by the dry-ice precipitation methoddescribed previously in step 1. A white product was afforded afterdrying in a desiccator (39 mg). ¹H-NMR (CDCl₃, 400 MHz) δ/ppm: 2.33 (s,6H), 2.68 (m, 2H), 3.14-3.22 (m, 4H), 3.37 (s, 3H), 3.5-3.8 (broad,PEG), 4.97 (br, 1H), 7.05 (d, 4H), 7.12 (d, 4H), 7.59 (d, 2H), 7.77 (d,2H)

Step 5: Preparation of Bis-PEG Aspartic DB Reagent 10

Aspartic PEG 10 kDa derivative 9 (50 mg) was dissolved in toluene (5 mL)for azeotropic distillation. Fresh N,N′-diisopropylcarbodiimide (DIPC)(2.4 mg, 2.79 μL) was added and the reaction mixture was allowed tostir, at room temperature for 30 min. O-(2-aminoethyl)-O′-methyl(polyethylene glycol) (PEG) 10 kDa (47.4 mg) was placed separately in aone neck 50 mL schlenk flask fitted with a septum and a magnetic stirbar. Azeotropic distillation was carried out using 5 mL of toluene, aspreviously described. Anhydrous DCM (5 mL) was added to the flask underargon atmosphere to dissolve the PEG completely. 4-Dimethylaminopyridine(DMAP) (0.58 mg) was added to the PEG solution. The activated asparticPEG derivative was added to the PEG solution drop-wise. The resultingsolution was allowed to stir for 48 h at room temperature under argonatmosphere. After this time, volatiles were removed by roto-evaporationand the crude product was placed under vacuum for 40 min. The crude wasre-dissolved in acetone for the purification by the dry-iceprecipitation method described in step 1. The solid obtained was driedin desiccator to afford a white-off product (79 mg). The same procedurewas applied for the preparation of bis-PEG aspartic DB reagent 2×20 kDa.¹H-NMR (CDCl₃, 400 MHz) δ/ppm: 2.34 (s, 6H), 2.68-2.93 (m, 2H),3.14-3.22 (m, 4H), 3.37 (s, 3H), 3.5-3.8 (broad, PEG), 4.94 (br, 1H),7.05 (d, 4H), 7.12 (d, 4H), 7.61 (d, 2H), 7.86 (d, 2H)

Step 6: Oxidation of Bis-Sulfide to Bis-Sulfone

Bis-PEG aspartic DB reagent 10 (20 mg) was dissolved in an aqueoussolution of 50% methanol (3 mL) in a 15 mL centrifuge tube. A magneticstirrer bar was added and while under agitation, OXONE® (potassiumperoxymonosulfate) was added to the solution. The reaction mixture wasallowed to stir over-night, at room temperature. After this time, thesolution was transferred to a 50 mL round-bottom flask and volatileswere removed by roto-evaporation. Purification of crude product wasachieved by dry-ice precipitation method described in step 1. The solidobtained was dried in a desiccator to afford a white-off product (16.2mg). ¹H-NMR (CDCl₃, 400 MHz) δ/ppm: 2.46 (s, 6H), 2.68 (m, 2H),3.14-3.22 (m, 4H), 3.37 (s, 3H), 3.5-3.8 (broad, PEG), 4.97 (br, 1H),7.35 (d, 4H), 7.59 (d, 6H), 7.77 (d, 2H).

EXAMPLE 6: PREPARATION OF PEG REAGENT 13

To a single neck round-bottomed flask containing a magnetic stirrer bar,was added bifurcated PEG amine 12 (100 mg, 40 kDa, JenKem Technology,code Y-NH2-40K, lot ZZ099P120) and toluene (8 mL). The resultinghomogeneous solution was evaporated under reduced pressure using arotary evaporator for 0.5 h to leave a solid residue. The residue wasdissolved in dichloromethane (3 mL), the flask was sealed with a septumand the mixture stirred under argon. To the solution was added compound4 (8.4 mg), the flask was resealed with a septum and the reaction wasstirred at room temperature for 48 h. The septum was removed and thevolatile portion was removed via evaporation under reduced pressureusing a rotary evaporator. Acetone (10 mL) was added to the residue andthe solid was dissolved with gentle warming (30° C.). The resultingsolution was filtered through non-absorbent cotton wool into a 15 mLFalcon tube. Cooling the solution in a dry-ice bath resulted in a thickprecipitate. Centrifugation (−9° C., 4000 rpm) for 30 min sedimented theprecipitate. The supernatant was decanted and the pellet was againdissolved in acetone (10 mL) at 30° C. Precipitation, sedimentation anddecanting were performed as previously described. A third cycle ofacetone precipitation and sedimentation was performed and afterdecanting the supernatant, the pellet was frozen at −80° C. and thendried to constant mass under high vacuum to give PEG reagent 13 as awhite solid (93 mg). ¹H NMR (CDCl₃): δ (ppm) 2.42 (s, 6H), 3.39 (s, 6H),3.46-3.84 (m), 4.34-4.38 (m, 1H), 7.05 (s, 1H), 7.36 (AA‘BB’, 4H), 7.64(AA‘BB’, 2H), 7.68 (AA‘BB’, 4H), 7.83 (AA‘BB’, 2H).

EXAMPLE 7: CONJUGATION OF PEG REAGENT 13 TO A FAB

A solution of a trastuzumab derived Fab (2.6 mg/mL, 2.5 mL) was bufferexchanged, using a PD-10 desalting column (GE Healthcare), into 50 mMsodium phosphate buffer, pH 7.4 (containing 150 mM NaCl and 20 mM EDTA).The resulting solution was then treated with DTT (1 M, 35 μL, 1 h, 4°C.) to reduce the interchain disulfide of the Fab. The disulfide reducedFab solution was then buffer exchanged into 50 mM sodium phosphatebuffer, pH 7.4, using two PD-desalting columns. Two aliquots of thesolution were then taken, each containing 1 mg of Fab. One aliquot wasstored at 4° C. and the other at room temperature (RT). A solution ofPEG reagent 13 was prepared in 50 mM sodium phosphate buffer, pH 7.4 (5mg/mL) and 175 μL of the solution was added to each of the reduced Fabsolutions. Each of the solutions was diluted, so that final Fabconcentration was 0.5 mg/mL. The reaction solutions were shaken gentlybefore being allowed to stand for 20 h. After 20 h, the samples (alongwith starting Fab and reduced Fab samples) were analysed by SDS-PAGE—thegels were stained with INSTANTBLUE™ and imaged using an IMAGEQUANT™ LAS4010 instrument. The results are shown in FIG. 6. In FIG. 6, the lanelabelled M shows Novex Protein Standards; lanes 1 and 4 show thestarting non-reduced Fab; lanes 2 and 5 show the reduced Fab; lane 3shows the 4° C. reaction mixture and lane 6 shows the room temperaturereaction mixture. From the SDS-PAGE analysis it can be seen that PEGreagent 13 conjugated successfully to the Fab fragment, the efficiencyof the conjugation at 4° C. was 24%, and at room temperature theefficiency was 40%.

We claim:
 1. A compound of general formula (I):

in which Q is:

in which each X independently represents a polymer chain; A represent agroup CO, CHOH, CH.NH₂, CH.NHR, CH.NR₂, CH.OR CH.O.C(O)R, CH.NHC(O)R, orCH.N(C(O)R)₂, in which each R represents a hydrogen atom or an alkyl,aryl or alkyl-aryl group; and either each of Pr and Pr² represents aseparate protein or peptide molecule or both of Pr¹ and Pr² togetherrepresent a single protein or peptide bonded at two separate points. 2.A compound as claimed in claim 1, having the general formula:

in which Q and A have the meanings given in claim 1, and Pr represents asingle protein or peptide bonded at two separate points.
 3. A compoundas claimed in claim 2, in which Pr represents a single protein orpeptide bonded via two sulfur atoms derived from a disulfide bond insaid protein or peptide, or via two histidine residues present in apolyhistidine tag attached to said protein or peptide.
 4. A compound asclaimed in claim 3, in which Pr represents an IgG Fab fragment, INF-α,IFN-β, or consensus IFN.
 5. A compound as claimed in claim 1, in whicheach X represents a poly(alkylene glycol), polyvinylpyrrolidone,polyacrylate, polymethacrylate, polyoxazoline, polyvinylalcohol,polyacrylamide, polymethacrylamide, HPMA copolymer, polyester,polyacetal, poly(ortho ester), polycarbonate, poly(imino carbonate),polyamide, or polysaccharide.
 6. A compound as claimed in claim 5, inwhich each X represent poly(ethylene glycol).
 7. A compound as claimedin claim 6, in which each X independently represents polyethylene glycolof the formula CH₃O—(CH₂CH₂O)_(m)— in which m is the number of ethyleneoxide units in X.
 8. A compound as claimed in claim 1, having theformula:

in which each X independently represents a polymer chain and each of Pr¹and Pr² represents a separate protein or peptide molecule.
 9. A compoundas claimed in claim 1, having the formula

in which each X independently represents a polymer chain and Prrepresents a single protein or peptide bonded at two separate points.10. A compound as claimed in claim 9, in which Pr represents a singleprotein or peptide bonded via two sulfur atoms derived from a disulfidebond in said protein or peptide, or via two histidine residues presentin a polyhistidine tag attached to said protein or peptide.
 11. Aprocess for the preparation of a compound as claimed in claim 1 in whichA represents a group CO, which comprises reacting a compound of generalformula (II):

in which Q has the meaning given in claim 1 and Z represents either—CH.(CH₂L)₂ or —C(CH₂L)(═CH₂), in which each L independently representsa leaving group; with a protein or a peptide.
 12. A process as claimedin claim 11, which comprises the additional step of reducing the ketogroup A in the resulting conjugate to form a compound of the formula Iin which A represents CHOH, CH.NH₂, CH.NHR, CH.NR₂, CH.OR CH.O.C(O)R,CH.NHC(O)R, or CH.N(C(O)R)₂, in which each R represents a hydrogen atomor an alkyl, aryl or alkyl-aryl group.
 13. A process as claimed in claim11, in which each L independently represents —SR, —SO₂R, —OSO₂R, —N⁺R₃,—N⁺HR₂, —N⁺H₂R, halogen, or —OØ, in which R represents a hydrogen atomor an alkyl, aryl or alkyl-aryl group, and Ø represents a substitutedaryl group containing at least one electron withdrawing substituent. 14.A process as claimed in claim 11, in which each L independentlyrepresents phenylsulfonyl or tosyl.
 15. A process as claimed in claim11, in which the compound of formula II has the formula:


16. A pharmaceutical composition comprising a compound as claimed inclaim 1, together with a pharmaceutically acceptable carrier.
 17. Apharmaceutical composition as claimed in claim 16, which contains afurther active ingredient.